key: cord-0764053-bc8ag40l
authors: Cao, Bin; Huang, Guo-Hong; Pu, Zeng-Hui; Qu, Jiu-Xin; Yu, Xiao-Min; Zhu, Zhen; Dong, Jian-Ping; Gao, Yan; Zhang, Yong-Xiang; Li, Xiao-Hui; Liu, Jian-Hua; Wang, Hong; Xu, Qian; Li, Hui; Xu, Wenbo; Wang, Chen
title: Emergence of Community-Acquired Adenovirus Type 55 as a Cause of Community-Onset Pneumonia
date: 2015-12-28
journal: Chest
DOI: 10.1378/chest.13-1186
sha: a19bb174be837fd7a2cbee20a107a21cf5c23465
doc_id: 764053
cord_uid: bc8ag40l

BACKGROUND: Since 2008, severe cases of emerging human adenovirus (HAdV) type 55 (HAdV-55) were reported sporadically in China. But no comparative studies had been conducted to discern the differences in epidemiologic and clinical abnormalities between HAdV-55 and other types (HAdV-7, HAdV-3, HAdV-14, HAdV-50, and HAdV-C). METHODS: A multicenter surveillance study for adult and adolescent community-acquired pneumonia (CAP) was conducted prospectively in Beijing and Yan Tai between November 2010 and April 2012. A standardized data form was used to record clinical information. The viral DNA extracted from the clinical samples or adenovirus viral isolates was sequenced. RESULTS: Among 969 cases, 48 (5%) were identified as adenovirus pneumonia. Six branches were clustered: HAdV-55 in 21, HAdV-7 in 11, HAdV-3 in nine, HAdV-14 in four, HAdV-50 in two, and HAdV-C in one. Most HAdV-55 cases were identified during February and March. All the hypervariable regions of the hexon genes of the 21 HAdV-55 strains were completely identical. Patients who had HAdV-55 were about 10 years older (P = .027) and had higher pneumonia severity index scores (P = .030) compared with those with other types (HAdV-7, HAdV-3, HAdV-14, HAdV-50, and HAdV-C). Systemic BP was also higher among patients in the HAdV-55 group (P = .006). Unilateral or bilateral consolidations were the most common radiologic findings in both patients with HAdV-55 and those with other types (57.9% vs 36%). More than one-half of the patients were admitted to hospital; oxygen therapy was given to 29.2% of the 48 patients, and two needed mechanical ventilation. CONCLUSIONS: HAdV-55 has established itself as a major pneumonia pathogen in the Chinese population, and further surveillance and monitoring of this agent as a cause of CAP is warranted.

The institutional review board of Beijing Chao-Yang Hospital approved the study (project approval number 10-KE-49). All patients gave their written informed consent.

Between November 2010 and April 2012, all adolescent and adult patients (aged 14 years or older) from 12 general hospitals who met the inclusion criteria of CAP were prospectively enrolled during daytime 7 days a week. 3 Patients with HIV infection or neutropenia, those receiving immunosuppressive chemotherapy or prednisone steroids equivalent to 15 mg/d for 30 days, pregnant or breast-feeding women, and those with known or suspected active TB were excluded.

Clinical information collected by investigators with a standardized data form included the following: age, sex, comorbidities, smoking history, vaccination against infl uenza and Streptococcus pneumoniae in the past year, symptoms (fever, cough, sputum, dyspnea, chest pain), GI symptoms (nausea, vomiting, diarrhea, and abdominal pain), and neurologic symptoms (headache, dizziness). Clinical signs (body temperature, heart rate, respiratory frequency, BP, and crackles) and treatments (antibiotics, antiviral therapy, or oxygen use) were also recorded. The pneumonia severity index (PSI) was used to assess the severity of illness on the day of enrollment. 17 Symptoms and signs of all patients were followed up, either during their hospitalization or after discharge, until all symptoms disappeared. For outpatients, the same information was gathered. All the information collected from the patients was input into a computerized database.

The nasal or throat swab specimens collected by the attending physicians were collected in 2-mL viral transport media, transported at 2°C to 8°C, and preserved at 2 80°C. The viral RNA was extracted from the clinical samples using a QIAamp RNA mini kit (QIAGEN). Following this, a commercially available Seeplex RV 15 ACE Detection kit (Seegene Inc), a multiplex, one-step, reverse transcriptase PCR, was used to screen for 15 different viruses as the cause of the respiratory illness. The kit included assays for adenovirus, infl uenza A and B viruses, human metapneumovirus, rhinovirus, respiratory syncytial virus (groups A and B), coronavirus (229E, NL63, OC43, and HKU1), parainfl uenza virus (type 1, 2, 3, 4), bocavirus, and enterovirus.

Blood cultures were performed for patients presenting with chills and shivering. If pleural fl uid and sputum samples were available, Gram stain and culture were performed. Urinary antigen tests for Legionella pneumophila and S pneumoniae (Binax) were also performed on all urine specimens. Acute sera (1-3 days after onset) and convalescent sera (2-4 weeks after onset) were collected for testing of the antibody for HAdV or other respiratory viruses.

Viral pneumonia was diagnosed based on one of the following criteria: (1) the presence of HAdV or other respiratory viruses detected in sputum or throat swab samples by molecular methods or (2) seroconversion, defi ned as a fourfold or greater increase in titers of antibodies to HAdV or other respiratory viruses.

Nasal or throat swab specimens were inoculated onto Hep-2 cells and cultured in a maintenance medium for detection of a respiratory tract infection in Shanxi Province, China, in 2006. 12 It exhibited a neutralizing antigen epitope of HAdV-11 and the pathogenic properties of HAdV-14. [13] [14] The whole-genome sequencing analysis showed that HAdV-55 had an HAdV-14 chassis with a partial HAdV-11 in the hexon gene. [15] [16] For this reason, it was renamed HAdV-55. 13 Our previous case series 11 indicated that HAdV-55 apparently emerged in Beijing . 12 Adenovirus 14 is an emerging agent of concern that has been causing outbreaks of pneumonia not just in China, but worldwide. Adenovirus 55, which is related to adenovirus 14, is now also emerging as an agent of concern. We investigated whether HAdV-55 has a different clinical profi le from the profi les of other adenovirus types circulating in China.

The Beijing Network for Adult Community-Acquired Pneumonia (BNACAP), which consists of 11 general hospitals from nine different districts in Beijing and one teaching hospital in Yan Tai, is a clinic-based, multicenter, prospective surveillance system for adults and adolescents with CAP. Yan Tai is a city by the sea in Shan Dong Province, located about 770 km southeast of Beijing.

Manuscript received May 19, 2013; revision accepted August 1, 2013 .

Between November 2010 and April 2012, 1,013 cases with CAP were enrolled in the BNACAP study. Forty-four cases were ruled out: In 30, no throat/nasal specimen was obtained, and clinical information was missing in 14. Therefore, 969 cases were available for the etiology study. Among them, 393 were positive for at least one pathogen: respiratory viruses in 262, Mycoplasma pneumoniae in 168, typical bacteria in 47, Mycobacterium tuberculosis in 15, and Legionella pneumoniae in four. Dual causes were found in 65 patients (e- Table 1 ). were HAdV-50, and one (2.1%) was HAdV-C. Most HAdVs were identifi ed in February and March. No adenovirus was found in November or December ( Fig 1 ) . The type distribution was similar between Beijing and Yan Tai City ( Table 1 ) . cytopathic effect (CPE). Cells were observed for CPE every 7 days. Cultures exhibiting adenovirus-like CPE were processed again to confi rm the presence of the virus.

Viral DNA was extracted from the clinical samples and adenovirus viral isolates using a QIAamp DNA mini kit (QIAGEN). The hexon and fi ber genes were both amplifi ed using primers described previously by Zhu et al. 12 PCR was performed with a 25-m L reaction mixture containing 2 m L of template DNA. Reaction conditions were determined as described previously by Zhu et al. 12 

The PCR products were purifi ed (QIAGEN) and sequenced by a dye terminator method (BigDye Terminator, version 3.1, cycle sequencing kit; Applied Biosystems) with an ABI Prism 3100 genetic analyzer (Applied Biosystems). Sequence data were stored as standard chromatogram format fi les (.abl) and were analyzed with Sequencher software ( 

Data analysis was performed using SPSS 15.0 (IBM). A twotailed independent-samples t test or a Mann-Whitney U test (in the case of nonnormal distributions) was used to compare continuous variables between the two groups. For the categorical data, univariate analysis was carried out using the x 2 test or Fisher exact test. Signifi cance was fi xed at P , .05. ( Table 2 ) .

Forty percent of the patients had bilateral involvement on chest radiography ( Table 2 ) . Consolidation, patchy infi ltrate, and ground-grass opacity were the most common fi ndings in pneumonia caused by HAdV. Patients infected by HAdV-55 presented consolidation more commonly than did those infected by other types (57.9% vs 36%) ( Fig 2 ) , but the difference was not signifi cant.

More than one-half of the patients were admitted to hospital, but there was no difference between HAdV-55 and other types ( Table 3 ) . No case was proved to have a coinfection with bacteria, but coinfections with other respiratory viruses (25%) or M pneumoniae (12.5%) were common. Oxygen therapy was given to 29.2% of the patients, and only two needed mechanical ventilation. Antibiotics were given to all the patients, but only four were prescribed antiviral drugs (all from Beijing Chao-Yang Hospital). The clinical outcomes, including duration of fever and other respiratory symptoms, length of stay in hospital, and hospitalization

The mean age of the 48 cases was 38 years; 12 of the 48 (25%) were aged . 50 years. Patients infected by HAdV-55 were about 10 years older than those infected by other types ( P 5 .027). Men predominated over women, with a sex ratio of about 2:1. More patients infected by HAdV-55 had underlying diseases (19.7% vs 3.7%), although the difference was not signifi cant ( Table 1 ) .

Most clinical symptoms and signs between patients infected by HAdV-55 and those infected by other types did not differ, except for PSI score and systemic BP; these were signifi cantly higher in patients infected by HAdV-55 ( P 5 .030 and P 5 .006, respectively) ( Table 1 ) .

There was no difference in laboratory fi ndings between HAdV-55-infected cases and those infected by other types ( Table 2 ) . Eight HAdV cases involved coinfections, including HAdV-55 with M pneumoniae in three, HAdV-55 with parainfl uenza virus 3 and infl uenza virus B in one, HAdV-7 with M pneumoniae in one, HAdV-2 with respiratory syncytial virus A in of HAdV strains were compared with the sequences of HAdV-B species in GenBank (e- Fig 1B) ; 21 of the 47 HAdV-Bs formed a dependent branch and revealed 100%, 96.7%, and 80% homologies with HAdV-55 (FJ643676), HAdV-11 (AF532578), and HAdV-14 (AY803294), respectively. Another phylogenetic tree was then conducted based on the partial fi ber gene of the 15 HAdVs, hexon genes which were homologous to HAdV-55 (e- Fig 1C) . The partial fi ber gene had 99.8% to 100%, 99.4% to 99.5%, and 94.2% to 94.3% nucleotide identity with HAdV-55 (FJ643676), expenses, were similar between HAdV-55 and other types ( Table 3 ) .

A phylogenetic analysis was conducted based on the hypervariable region of the hexon gene to demonstrate the genetic relationship between HAdVs strains and the other seven HAdVs species (A-G); 47 of 48 strains belonged to HAdV species B and only one was HAdV-C (e- Fig 1A) . Further partial hexon gene Data are presented as mean Ϯ SD, No. (%), or median (range). ALB 5 albumin; ALT 5 alanine aminotransferase; AST 5 aspartate aminotransferase; CK 5 creatine kinase; Cr 5 creatinine; CRP 5 C reactive protein; ESR 5 erythrocyte sedimentation rate; LDH 5 lactate dehydrogenase; PCT 5 procalcitonin; Tbil 5 total bilirubin. See Table 1 legend for expansion of other abbreviations . a n 5 27. b n 5 23. HAdV has been recognized as an important viral cause of ARDS. The HAdV types most frequently associated with ARDSs include subspecies B1 HAdV-3, HAdV-7, and HAdV-21 and species E HAdV-4. The association of subspecies B2 HAdV (HAdV-11, HAdV-14, HAdV-34, HAdV-35) infection with ARDS has been rarely reported historically, with some of that documentation cover ing military trainees. 15, 18, 19 In China, HAdV-3 and HAdV-7 were the most common types of pathogens. [20] [21] [22] HAdV-11a associated with ARDSs can be traced back to the 1980s 23 and it reemerged as an ARDS pathogen in 2006. 12, 16 HAdV-55 (formerly known as HAdV-11a) was renamed HAdV-55 based on complete genomic sequence data, 13 which clearly showed that HAdV-55 was a recombinant between the HAdV-11 and HAdV-14 ancestral strains.

Adenovirus 14 is an emerging agent of concern that has been causing outbreaks of pneumonia not just in China but worldwide. 24, 25 Tate et al 26 reported an outbreak of severe respiratory disease associated with HAdV-14 in a US Air Force training facility. Five hundred fi fty-one of 1,147 trainees (48%) with febrile respiratory illness were infected with HAdV-14; 23 train ees were hospitalized with pneumonia; four of those required admission to an ICU, and one died. Subsequently, HAdV-14 (AY803294), and HAdV-11 (AF532578), respectively. (The amplifi cation or sequencing of the other six strains failed, and the real-time PCR indicated that their hexon genes were HAdV-14 and their fi ber genes were HAdV-14 [data not show]).

All the hypervariable regions of the hexon genes (18281-19247nt, 967bp) of the 21 HAdV-55 strains were completely identical. Two of the 15 partial fi ber genes (30689-31818nt, 1130bp) had one nucleotide difference from the others. The nucleotide sequences data from these 48 HAdVs were submitted to GenBank, and accession numbers were allocated as KC510700 to KC510747 (Hexon genes) and KC510748 to KC510762 (fi ber genes).

To our knowledge, this study is the fi rst large cohort on the epidemiology and clinical features of CAP associated with HAdV-55, the emerging pathogen among immunocompetent adolescents and adults. Our data showed clearly that HAdV-55 has established itself as a major pneumonia pathogen in the Chinese population and that further surveillance and monitoring of this agent as a cause of CAP is warranted. cause determination and a good surveillance system are important.

Author contributions : Drs Cao and Wang had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Dr Cao: contributed to the design of the study, care of the adenovirus pneumonia cases, data gathering, analysis of clinical data, writing of the manuscript, and the decision to publish. Dr Huang: contributed to the PCR analysis and genotyping and writing of the manuscript. Dr Pu: contributed to data gathering and manuscript revision. Dr Qu: contributed to the care of the adenovirus pneumonia cases, data gathering, clinical specimen collection, PCR analysis, and manuscript revision. Dr Yu: contributed to the care of the adenovirus pneumonia cases, data gathering, analysis of clinical data, and manuscript revision. outbreaks associated with HAdV-14 were reported in other states, such as Oregon, Alaska, and so forth. [27] [28] Today, adenovirus 55, which is related to adenovirus 14, is also emerging as an agent of concern. Because of the absence of cases caused by other HAdV types, Vento et al 29 could only compare pneumonia caused by HAdV-14 with HAdV-14-negative cases. Our study had the advantage of being able to compare the differences in clinical, laboratory, or radiographic abnormalities caused by HAdV-55 and other types of pathogens. We proved that patients with diseases due to HAdV-55 were about 10 years older ( P 5 .027) and had higher PSI scores ( P 5 .030) Our study has several limitations. First, our case series mainly represents the relatively mild and moderate end of the disease. We believe a wider surveillance study is needed to evaluate the spectrum of the disease caused by this emerging pathogen in an affected area in China. In addition, virus isolates were typed only by amplifi cation and sequencing of the hexon gene, and no seroneutralization was performed. More laboratory tests should be carried out to understand the genomics and pathogenic characteristics.

In conclusion, our data provide new insight into the epidemiology of HAdV-55 infection in China. Patients with HAdV-55 infection were about 10 years older and had higher PSI scores than did patients infected by other types (HAdV-3, HAdV-7, HAdV-14) . Furthermore, because it is diffi cult to discern HAdV pneu mo nia from clinical symptoms and signs, viral Table 1 legend for expansion of other abbreviations. a Three of the patients were prescribed acyclovir and the fourth was given ribavirin. All the patients were from Beijing Chao-Yang Hospital.

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Outbreak of severe disease associated with emergent human adenovirus serotypes 14 at a US air force training facility in 2007

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Financial/nonfi nancial disclosures: The authors have reported to CHEST that no potential confl icts of interest exist with any companies/organizations whose products or services may be discussed in this article .

The sponsors had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript. Other contributions: We thank all the doctors who have joined the BNACAP network but are not listed in the authorship. We also thank Yingmei Liu, MD, and Zhenjia Liu, MD, for their contribution to the specimen collection and PCR analysis. Additional information: The e-Figure and e-Table can be found in the "Supplemental Materials" area of the online article.